Abstract Addiction is a learning and memory disorder, wherein conditioned responses to seek drug lead to chronic relapse. These conditioned responses can be triggered by exposure to reminder cues associated with the drug, and they can be inhibited through a process called extinction. The prefrontal cortex, especially the infralimbic(IL)subregioninrodents,iscommonlyrecruitedduringextinctionlearningtoexertinhibitorycontrol overbehavior.Thisistrueforanumberofbehaviors,includingconditionedcocaineseeking,foodseeking,and fear.However,preliminarydatasuggestthatthisregionisnotrecruitedduringextinctionofheroinseeking,an observationconsistentwithnumerousreportssuggestingtheroleofILcortexmaybefundamentallydifferent for heroin seeking. Alternatively, there may be distinct neuronal ensembles within IL that drive versus inhibit drug seeking. The anatomical connections of IL include three major efferent targets known to regulate drug seeking, namely the lateral hypothalamus, the nucleus accumbens shell, and the basolateral amygdala. We proposethatILneuronalensemblesprojectingtodistincttargetswilldriveversusinhibitheroinversuscocaine seeking,respectively.Aim1ofthisNIDAR21proposalwilldeterminewhetherfunctionalneuronalensembles existwithinILthatarecapableofdrivingversusinhibitingheroinandcocaineseeking.Fos-activatedneurons will be tagged with designer receptors (DREADDs) under control of a tetracycline-dependent promoter, and subsequently reactivated using the designer drug, clozapine-N-oxide. This chemogenetic form of memory retrievalwillbeusedtoexaminetheroleoftheoriginaldrug-cuememoryversusextinctionmemoryonrelapse. Aim 2 of this proposal will examine the efferent targets of these ensembles using the retrograde tracer CAV- GFP.TheseexperimentswilldeterminewhethertheinhibitoryfunctionofIListrulyabsentforheroinseeking, or whether it has been overlooked due to the use of inadequate methods targeting all IL neurons indiscriminately.Furthermore,thisworkwillprovidenewinsightintothespecificneuroanatomyofdrugmemory engramsandtheextenttowhichtheycanbeexploitedtoreducerelapse.